John C. Clapham

Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean

Uncoupling protein-3 (UCP-3) is a recently identified member of the mitochondrial transporter superfamily that is expressed predominantly in skeletal muscle. However, its close relative UCP-1 is expressed exclusively in brown adipose tissue, a tissue whose main function is fat combustion and thermogenesis. Studies on the expression of UCP-3 in animals and humans in different physiological situations support a role for UCP-3 in energy balance and lipid metabolism. However, direct evidence for these roles is lacking. Here we describe the creation of transgenic mice that overexpress human UCP-3 in skeletal muscle. These mice are hyperphagic but weigh less than their wild-type littermates. Magnetic resonance imaging shows a striking reduction in adipose tissue mass. The mice also exhibit lower fasting plasma glucose and insulin levels and an increased glucose clearance rate. This provides evidence that skeletal muscle UCP-3 has the potential to influence metabolic rate and glucose homeostasis in the whole animal.

Oxidative damage and phospholipid fatty acyl composition in skeletal muscle mitochondria from mice underexpressing or overexpressing uncoupling protein 3

Five markers of different kinds of oxidative damage to proteins [glutamic semialdehyde, aminoadipic semialdehyde, N (epsilon)-(carboxymethyl)lysine, N (epsilon)-(carboxyethyl)lysine and N (epsilon)-(malondialdehyde)lysine] and phospholipid fatty acyl composition were identified and measured in skeletal muscle mitochondria isolated from mice genetically engineered to underexpress or overexpress uncoupling protein 3 (UCP3). Mitochondria from UCP3-underexpressing mice had significantly higher levels of oxidative damage than wild-type controls, suggesting that UCP3 functions in vivo as part of the antioxidant defences of the cell, but mitochondria from UCP3-overexpressing mice had unaltered oxidative damage, suggesting that mild uncoupling in vivo beyond the normal basal uncoupling provides little protection against oxidative stress. Mitochondria from UCP3-underexpressing mice showed little change, but mitochondria from UCP3-overexpressing mice showed marked changes in mitochondrial phospholipid fatty acyl composition. These changes were very similar to those previously found to correlate with basal proton conductance in mitochondria from a range of species and treatments, suggesting that high protein expression, or some secondary result of uncoupling, may cause the observed correlation between basal proton conductance and phospholipid fatty acyl composition.

Set points, settling points and some alternative models: Theoretical options to understand how genes and environments combine to regulate body adiposity

The close correspondence between energy intake and expenditure over prolonged time periods, coupled with an apparent protection of the level of body adiposity in the face of perturbations of energy balance, has led to the idea that body fatness is regulated via mechanisms that control intake and energy expenditure. Two models have dominated the discussion of how this regulation might take place. The set point model is rooted in physiology, genetics and molecular biology, and suggests that there is an active feedback mechanism linking adipose tissue (stored energy) to intake and expenditure via a set point, presumably encoded in the brain. This model is consistent with many of the biological aspects of energy balance, but struggles to explain the many significant environmental and social influences on obesity, food intake and physical activity. More importantly, the set point model does not effectively explain the ‘obesity epidemic’ – the large increase in body weight and adiposity of a large proportion of individuals in many countries since the 1980s. An alternative model, called the settling point model, is based on the idea that there is passive feedback between the size of the body stores and aspects of expenditure. This model accommodates many of the social and environmental characteristics of energy balance, but struggles to explain some of the biological and genetic aspects. The shortcomings of these two models reflect their failure to address the gene-by-environment interactions that dominate the regulation of body weight. We discuss two additional models – the general intake model and the dual intervention point model – that address this issue and might offer better ways to understand how body fatness is controlled.

Anorectic, thermogenic and anti-obesity activity of a selective orexin-1 receptor antagonist in ob/ob mice

A single dose of the orexin-1 (OX1) receptor antagonist 1-(2-methylbenzoxazol-6-yl)-3-[1,5] naphthyridin-4-yl urea hydrochloride (SB-334867-A) reduces orexin-A-induced feeding and natural feeding in Sprague Dawley rats. In this study, the anti-obesity effects of SB-334867-A were determined in genetically obese (ob/ob) mice dosed with SB-334867-A (30 mg/kg, i.p.) once daily for 7 days, and then twice daily for a further 7 days. SB-334867-A reduced cumulative food intake and body weight gain over 14 days. Total fat mass gain, determined by Dual Emission X-ray Absorptiometry, was reduced, while gain in fat-free mass was unchanged. Fasting (5 h) blood glucose was also reduced at the end of the study, with a trend to reduced plasma insulin. Interscapular brown adipose tissue (BAT) weight was reduced, the tissue was noticeably darker in colour and quantitative PCR (TaqMan) analysis of this tissue showed a trend to an increase in uncoupling protein-1 mRNA expression, suggesting that SB-334867-A might stimulate thermogenesis. This was confirmed in a separate study in which a single dose of SB-334867-A (30 mg/kg, i.p.) increased metabolic rate over 4 h in ob/ob mice. OX1 receptor mRNA was detected in BAT, and its expression was increased by 58% by treatment with SB-334867-A. This is the first demonstration that OX1 receptor antagonists have potential as both anti-obesity and anti-diabetic agents.

Anti-obesity drugs: a critical review of current therapies and future opportunities

The last 25 years have seen a great increase in the incidence of obesity, both in the Western world and in developing third world countries. Despite the seeming inexorable progression of this disease, there have been limited advances in the pharmacotherapy of this condition. Of the newest introductions to the obesity drug portfolio, orlistat, which acts to prevent dietary fat absorption, and sibutramine, which seems to affect both arms of the energy balance equation, were the first new chemical entities to be introduced for the treatment of obesity in 30 years. In this article, we review these and other agents available in various countries for the treatment of obesity. Perhaps more importantly, we have focussed on areas of potential productivity in the future. The huge recent increase in our knowledge in this area has largely stemmed from discovery research at the genomics level. Over the last 5 or so years, this impetus in obesity research has provided us with exciting new drug targets involved in the regulation of feeding behaviour and cellular mechanisms involved in energy expenditure. Compared with the last 25 years, the future offers more hope.

BRL 34915, a novel antihypertensive agent: comparison of effects on blood pressure and other haemodynamic parameters with those of nifedipine in animal models.

The effects of BRL 34915, (+/-) 6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1-pyrrolidyl)-2H-b enzo [b]-pyran-3-ol, on blood pressure and other haemodynamic parameters in animals have been investigated in comparison with those of nifedipine. In conscious spontaneously hypertensive rats and renal hypertensive cats and dogs the oral doses of BRL 34915 lowering blood pressure are 10 to 30 times lower than those of nifedipine. Tachycardia evoked by BRL 34915 tends to be less than that produced by nifedipine in the cat and of similar magnitude in the dog. The antihypertensive response to BRL 34915 in these models is reproducible on repeat once daily dosing without rebound hypertension on cessation of dosing. In studies using electromagnetic flow probes to measure regional blood flow in anaesthetised cats the intravenous administration of BRL 34915, unlike that of nifedipine, markedly increases renal blood flow yet BRL 34915 lacks the marked effect of nifedipine on femoral blood flow. BRL 34915, a compound structurally unrelated to existing cardiovascular drugs, is a potent new antihypertensive agent having an interesting profile of activity that renders this compound of clinical interest.

Liver-directed overexpression of mitochondrial glycerol-3-phosphate acyltransferase results in hepatic steatosis, increased triacylglycerol secretion and reduced fatty acid oxidation

Glycerol‐3‐phosphate acyltransferase (GPAT) catalyzes the first committed step in triacylglycerol (TAG) and phospholipid biosynthesis. GPAT activity has been identified in both ER and mitochondrial subcellular fractions. The ER activity dominates in most tissues except in liver, where the mitochondrial isoform (mtGPAT) can constitute up to 50% of the total activity. To study the in vivo effects of hepatic mtGPAT overexpression, mice were transduced with adenoviruses expressing either murine mtGPAT or a catalytically inactive variant of the enzyme. Overexpressing mtGPAT resulted in massive 12‐and 7‐fold accumulation of liver TAG and diacylglycerol, respectively but had no effect on phospholipid or cholesterol ester content. Histological analysis showed extensive lipid accumulation in hepatocytes. Furthermore, mtGPAT transduction markedly increased adipocyte differentiation‐related protein and stearoyl‐CoA desaturase‐1 (SCD‐1) in the liver. In line with increased SCD‐1 expression, 18:1 and 16:1 in the hepatic TAG fraction increased. In addition, mtGPAT overexpression decreased ex vivo fatty acid oxidation, increased liver TAG secretion rate 2‐fold, and increased plasma TAG and cholesterol levels. These results support the hypothesis that increased hepatic mtGPAT activity associated with obesity and insulin resistance contributes to increased TAG biosynthesis and inhibition of fatty acid oxidation, responses that would promote hepatic steatosis and dyslipidemia.—Lindén, D., William‐Olsson, L., Ahnmark, A., Ekroos, K., Hallberg, C., Sjögren, H. P., Becker, B., Svensson, L., Clapham, J. C., Oscarsson, J., Schreyer, S. Liver‐directed overexpression of mitochondrial glycerol‐3‐phosphate acyltransferase results in hepatic steatosis, increased triacylglycerol secretion and reduced fatty acid oxidation. FASEB J. 20, 434–443 (2006)

DPP-IV inhibition enhances the antilipolytic action of NPY in human adipose tissue

Context:  Dipeptidyl peptidase IV (DPP‐IV) inactivates the incretin hormone glucagon‐like peptide. It can also affect the orexigenic hormone neuropeptide Y (NPY1–36) which is truncated by DPP‐IV to NPY3–36, as a consequence NPY’s affinity changes from receptor Y1, which mediates the antilipolytic function of NPY, to other NPY receptors. Little is known whether DPP‐IV inhibitors for the treatment of type 2 diabetic (T2DM) patients could influence these pathways.

Increased fatty acid oxidation in transgenic mice overexpressing UCP3 in skeletal muscle.

Aim:  To determine the rates of substrate oxidation by skeletal muscle in vitro as well as tissue‐specific glucose uptake in vivo in transgenic mice overexpressing uncoupling protein‐3 (UCP3) in skeletal muscle.

Thermogenic and metabolic antiobesity drugs: rationale and opportunities

Antiobesity drugs that target peripheral metabolism may avoid some of the problems that have been encountered with centrally acting anorectic drugs. Moreover, if they cause weight loss by increasing fat oxidation, they not only address a cause of obesity but also should promote loss of fat rather than lean tissue and improve insulin sensitivity. Weight loss may be slow but more sustained than with anorectic drugs, and thermogenesis may be insufficient to cause any discomfort. Some thermogenic approaches are the activation of adrenergic, thyroid hormone or growth hormone receptors and the inhibition of glucocorticoid receptors; the modulation of transcription factors [e.g. peroxisome proliferator‐activated receptor δ (PPARδ) activators] or enzymes [e.g. glutamine fructose‐6‐phosphate amidotransferase (GFAT) inhibitors] that promote mitochondrial biogenesis, and the modulation of transcription factors (PPAR α activators) or enzymes (AMP‐activated protein kinase) that promote fatty acid oxidation. More surprisingly, studies on genetically modified animals and with enzyme inhibitors suggest that inhibitors of fatty acid synthesis [e.g. ATP citrate lyase, fatty acid synthase, acetyl‐CoA carboxylase (ACC)], fatty acid interconversion [stearoyl‐CoA desaturase (SCD)] and triglyceride synthesis (e.g. acyl‐CoA : diacylglycerol acyltransferase) may all be thermogenic. Some targets have been validated only by deleting genes in the whole animal. In these cases, it is possible that deletion of the protein in the brain is responsible for the effect on adiposity, and therefore a centrally penetrant drug would be required. Moreover, whilst a genetically modified mouse may display resistance to obesity in response to a high fat diet, it requires a tool compound to demonstrate that a drug might actually cause weight loss. Even then, it is possible that differences between rodents and humans, such as the greater thermogenic capacity of rodents, may give a misleading impression of the potential of a drug.